Process for preparing 9α-OH BN acid methyl ester

ABSTRACT

Valuable steroid intermediates, 9α-hydroxyandrost-4-ene-17β-ol-3-one (9α-OH testosterone), 9α-hydroxy-3-ketobisnorchol-4-en-22-ol (9α-OH BN alcohol) and 9α-hydroxy-3-ketobisnorchol-4-en-22-oic methyl ester (9α-OH BN acid methyl ester), prepared by microbiological conversion of steroids having 17-alkyl side chains of 8 to 10 carbons.

This is a division of application Ser. No. 844,366, filed Oct. 21, 1977.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 4,029,549 discloses and claims a steroid conversionprocess for making 9α-hydroxy-3-ketobisnorchol-4-en-22-oic acid (9α-OHBN acid). The process can be conducted using a mutant of a variety ofsteroid degrading microorganisms. The mutation process to prepare themutants is disclosed in the patent. Specifically exemplified is the useof Mycobacterium fortuitum NRRL B-8119.

U.S. Pat. No. 4,035,236 discloses and claims a process for preparing9α-hydroxyandrostenedione (9α-OH AD). This compound is also produced bythe process as disclosed in U.S. Pat. No. 4,029,549.

The presence of additional compounds in the fermentation beers disclosedin the above patents was previously recognized, but the identity of thecompounds was not known prior to the date of the subject invention.These additional compounds were subsequently shown by advancedidentification techniques to be useful steroid intermediates asdisclosed herein. Of these compounds, 9α-OH testosterone is a knowncompound, whereas the others are novel.

BRIEF SUMMARY OF THE INVENTION

9α-OH testosterone, 9α-OH BN alcohol and 9α-OH BN acid methyl ester areproduced in a fermentation process using the microorganism Mycobacteriumfortuitum NRRL B-8119. This organism is disclosed and characterized inU.S. Pat. No. 4,029,549. In addition to the characteristics given insaid patent, this microorganism is further characterized by its abilityto accumulate the compounds disclosed herein under fermentationconditions, also as disclosed herein. Other mutants of Mycobacterium, aswell as mutants from the genera of microorganisms disclosed in U.S. Pat.No. 4,029,549, can be used in the subject invention. Examples ofsuitable steroid substrates are sitosterol, cholesterol, stigmasterol,campesterol, and like steroids with 17-alkyl side chains of from 8 to 10carbon atoms, inclusive. These steroid substrates can be in either thepure or crude form.

DETAILED DESCRIPTION OF THE INVENTION

The microorganisms which can be used to produce the compounds of thesubject invention are the same as disclosed in U.S. Pat. No. 4,029,549.The microorganism specifically exemplified as Mycobacterium fortuitum,NRRL B-8119. A subculture of this microorganism is freely available fromthe depository at the Northern Regional Research Laboratory, U.S.Department of Agriculture, Peoria, Ill., U.S.A., by request madethereto. It should be understood that the availability of the culturedoes not constitute a license to practice the subject invention inderogation of patent rights granted with the subject instrument bygovernmental action.

The transformation process of the subject invention is also as disclosedin U.S. Pat. No. 4,029,549.

Also, the procedure for the preparation of Mycobacterium fortuitum NRRLB-8119 is as disclosed in U.S. Pat. No. 4,029,549. This process can alsobe used to prepare mutants of other genera of microorganisms asdisclosed in U.S. Pat. No. 4,029,549 and herein.

The isolation of the products of the subject invention from thefermentation broth is accomplished by first removing the major productsof the sterol conversions, i.e., 9α-OH AD and 9α-OH BN acid. These majorproducts are recovered from the fermentation beer by first extractingthe fermentation beer with a water-immiscible organic solvent forsteroids. Suitable solvents are methylene chloride (preferred),chloroform, carbon tetrachloride, ethylene chloride, trichloroethylene,ether, amyl acetate, benzene and the like.

Alternatively, the fermentation liquor and cells can be first separatedby conventional methods, e.g., filtration or centrifugation, and thenseparately extracted with suitable solvents. The cells can be extractedwith either water-miscible or water-immiscible solvents. Thefermentation liquor, freed of cells, can be extracted withwater-immiscible solvents.

The extract from the fermentation beer is dried. The resulting solidsare taken up in chloroform and sufficient methanol is added toprecipitate residual sterols which are then filtered off. The filtrateis dried and the residue dissolved in hot acetone. Upon cooling andsubsequent addition of cyclohexane most of the 9α-OH AD is precipitatedand recovered by filtration. The filtrate is then dried and the residuedissolved in chloroform and extracted with a saturated sodiumbicarbonate solution to remove 9α-OH BN acid. The individual componentsremaining in the chloroform after the bicarbonate extraction areseparated by chromatography on a silica gel column, eluting withchloroform-methanol (98:2).

The first compound to elute is the methyl ester of 9α-OH BN acid. Thesecond compound to elute is residual 9α-OH AD which remains soluble inthe acetone-cyclohexane solution described above. The third compound is9α-OH BN alcohol. The next compound to elute from the column is 9α-OHtestosterone.

The compounds of the subject invention are valuable as intermediates inthe manufacture of steroids. For example, 9α-OH BN acid methyl ester canbe converted to 9(11)-dehydro BN acid by treatment with N-bromoacetamideand sulfur dioxide in pyridine, as disclosed in British Patent No.869,815, followed by hydrolysis to generate the 22-carboxyl.9(11)-Dehydro BN acid can be converted to 9(11)-dehydroprogesterone by,for example, the method described in Ber. 88: 883 (1955), andsubsequently to 11β-hydroxyprogesterone as described in JACS 88: 3016(1966). Treatment of 11β-hydroxyprogesterone with chromic acid yields11-ketoprogesterone which is a known intermediate in the synthesis ofcortisol acetate, a major and highly active cortical steroid [see, forexample, Fieser and Fieser, Steroids, page 676, Reinhold (1959)].

9α-OH BN alcohol can be readily converted to 9α-OH BN acid by chromicacid oxidation, then to 9α-OH BN acid methyl ester by treatment withdiazomethane and subsequently to 11-ketoprogesterone as described above.

9α-Hydroxy-11-unsubstituted steroids of the androstane series can alsoeasily be dehydrated to the valuable 9(11)-dehydro steroids inaccordance with methods known in the art, e.g., with thionyl chloride inthe presence of pyridine. The 9(11)-dehydro compounds thus obtained areknown intermediates in the production of highly active compounds. Forexample, the 9(11)-dehydro steroids can be converted to thecorresponding 9α-halo-11β-hydroxy compounds in accordance withprocedures known in the art, e.g., U.S. Pat. No. 2,852,511 for thepreparation of 9α-halo-hydrocortisone. Also, 9α-hydroxy compounds of theandrostane series are useful as antiandrogenic, antiestrogenic andantifertility agents.

The following examples are illustrative of the process and products ofthe subject invention but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Preparation of Mutant M. fortuitum NRRL B-8119 From M.fortuitum ATCC 6842. (a) Nitrosoquanidine Mutagenesis

Cells of M. fortuitum ATCC 6842 are grown at 28° C. in the followingsterile seed medium:

Nutrient Broth (Difco)--8 g/liter

Yeast Extract--1 g/liter

Sodium Propionate--0.5 g/liter

Distilled Water, q.s.--1 liter

The pH is adjusted to 7.0 with 1 N NaOH prior to sterilization at 121°C. for 20 minutes.

The cells are grown to a density of about 5×10⁸ per ml, pelleted bycentrifugation, and then washed with an equal volume of sterile 0.1 Msodium citrate, pH 5.6. Washed cells are resuspended in the same volumeof citrate buffer, a sample removed for titering (cell count), andnitrosoguanidine added to a final concentration of 50 μg/ml. The cellsuspension is incubated at 37° C. in a water bath for 30 minutes, afterwhich a sample is again removed for titering and the remaindercentrifuged down and washed with an equal volume of sterile 0.1 Mpotassium phosphate, pH 7.0. Finally, the cells are resuspended in asterile minimal salts medium, minus a carbon source, consisting of thefollowing:

NH₄ NO₃ --1.0 g/liter

K₂ HPO₄ --0.25 g/liter

MgSO₄.7H₂ O--0.25 g/liter

NaCl--0.005 g/liter

FeSO₄.7H₂ O--0.001 g/liter

Distilled Water, q.s.--1 liter

The pH is adjusted to 7.0 with 1 N HCl prior to sterilization at 121° C.for 20 minutes. The cells are then plated out to select for mutants.

(b) Selection And Isolation of Mutant M. fortuitum NRRL B-8119

Mutagenized cells, as described above, are diluted and spread ontoplates containing a medium consisting of the following (modified fromFraser and Jerrel. 1963. J. Biol. Chem. 205: 291-295):

Glycerol--10.0 g/liter

K₂ HPO₄ --0.5 g/liter

NH₄ Cl--1.5 g/liter

MgSO₄.7H₂ O--0.5 g/liter

FeCl₃.6H₂ O--0.05 g/liter

Distilled Water, q.s.--1 liter

Agar (15 g/liter) is added, and the medium is autoclaved at 121° C. for30 minutes and then poured into sterile Petri plates.

Growth on this medium eliminates most nutritional auxotrophes producedby the mutagensis procedure, e.g. cultures that require vitamins, growthfactors, etc. in order to grow on chemically defined medium areeliminated. After incubation at 28° C. for about 7 days, the resultingcolonies are replicated to test plates suitable for selecting mutantsand then back onto control plates containing the glycerol-based medium.The test plates are prepared as described by Peterson, G. E., H. L.Lewis and J. R. David. 1962. "Preparation of uniform dispersions ofcholesterol and other water-insoluble carbon sources in agar media." J.Lipid Research 3: 275-276. The minimal salts medium in these plates isas described above in section (a) of Example 1. Agar (15 g/liter), andan appropriate carbon source (1.0 g/liter), such as sitosterol orandrostenedione (AD), are added and the resulting suspension autoclavedfor 30 minutes at 121° C. The sterile, hot mixture is then poured into asterile blender vessel, blended for several minutes, and then pouredinto sterile Petri plates. Foaming tends to be a problem in thisprocedure but can be reduced by blending when the mixture is hot and byflaming the surface of the molten agar plates. In this manner uniformdispersions of water-insoluble carbon sources are obtained whichfacilitates the preparation of very homogenous but opaque agar plates.

Colonies which grew on the control plates, but not on test platescontaining AD as the sole carbon source, are purified by streaking ontonutrient agar plates. After growth at 28° C., individual clones arepicked from the nutrient agar plates with sterile toothpicks andretested by inoculating gridded plates containing AD as the carbonsource. Purified isolates which exhibit a phenotype different from theparental culture are then evaluated in shake flasks.

(c) Shake Flask Evaluation

Shake flasks (500 ml) contain 100 ml of biotransformation mediumconsisting of the following ingredients:

Glycerol--10.0 g/liter

K₂ HPO₄ --0.5 g/liter

NH₄ Cl--1.5 g/liter

MgSO₄.7H₂ O--0.5 g/liter

FeCl₃.6H₂ O--0.05 g/liter

Distilled Water, q.s.--1 liter

Soyflour (1 g/liter) is blended into the medium and then sitosterol (10g/liter) is also blended into the medium. After the flasks areautoclaved for 30 minutes at 121° C., they are cooled to 28° C. and theninoculated with 10 ml of seed growth prepared as follows:

The purified isolates from part (b) are grown on agar slants at 28° C. Aloop of cells taken from a slant is used to inoculate a 500-ml flaskcontaining 100 ml of sterile seed medium consisting of the followingingredients:

Nutrient Broth (Difco)--8 g/liter

Yeast Extract--1 g/liter

Glycerol--5 g/liter

Distilled Water, q.s.--1 liter

The pH is adjusted to 7.0 with 1 N NaOH prior to autoclaving the flasksat 121° C. for 20 minutes. The seed flasks are incubated at 28° C. for72 hours.

As disclosed above, 10 ml of seed growth is then used to inoculate each500-ml flask containing 100 ml of sterile transformation medium. Theflasks are then incubated at 28° C. to 30° C. on a rotary shaker andsampled at various intervals. Ten ml samples are removed and extractedby shaking with 3 volumes of methylene chloride, Portions of theextracts are analyzed by thin layer chromatography (tlc) using silicagel and the solvent system described above, i.e., 2:3 (by volume) ethylacetate-cyclohexane, and by gas-liquid chromatography. Evidence of thepresence of 9α-OH AD confirms the selective degradation of sitosterol bythe novel mutant produced from the parent M. fortuitum ATCC 6842.

EXAMPLE 2

To a medium consisting of 1.0 part of glycerol, 0.15 part of ammoniumchloride, 0.05 part of magnesium sulfate heptahydrate, 0.05 part ofdipotassium hydrogen phosphate, 0.005 part of ferric chloridehexahydrate, and 100 parts of distilled water is added 0.1 part ofsoyflour and 1.0 part of sitosterols, N.F. The resultant mixture issterilized by heating 30 minutes at 121° C., whereupon it is cooled to30° C. and then inoculated with 10 parts of a seed culture of the mutantMycobacterium fortuitum NRRL B-8119, prepared as described in Example1(c). The inoculated mixture is incubated at 30° C. for 336 hours withagitation to promote submerged growth. Following incubation, the mixtureis extracted with methylene chloride. The extract is filtered throughdiatomaceous earth and the filtrate is vacuum distilled to dryness. Theresidue is taken up in 10% by chloroform in methanol and thenconcentrated with nitrogen on a steam bath until crystals appear. Thesolution is then cooled to room temperature and filtered to remove theprecipitated sitosterols. From the supernatant, on evaporation ofsolvent, good yields of 9α-OH testosterone, 9α-OH BN alcohol and 9α-OHBN acid methyl ester, as well as 9α-OH AD and 9α-OH BN acid areobtained.

EXAMPLE 3

By substituting cholesterol for sitosterol in Example 2 there areobtained the compounds produced in Example 2.

EXAMPLE 4

By substituting stigmasterol for sitosterol in Example 2 there areobtained the compounds produced in Example 2.

EXAMPLE 5

By substituting campesterol for sitosterol in Example 2 there areobtained the compounds produced in Example 2.

EXAMPLE 6

By adding a combination of any of the steroids in Examples 2-5, inaddition to sitosterol, or in place of sitosterol, in Example 2 thereare obtained the compounds produced in Example 2.

EXAMPLE 7

The products produced in Example 2 can be isolated as separate entitiesin the essentially pure form by the following procedure. The supernatantof Example 2, containing the products produced in the fermentation, isdried and the residue dissolved in hot acetone. Upon cooling andsubsequent addition of cyclohexane most of the major product, 9α-OH AD,is precipitated and recovered by filtration. The filtrate is then driedand the residue dissolved in chloroform and extracted with a saturatedsodium bicarbonate solution to remove 9α-OH BN acid. The individualcomponents remaining in the chloroform after the bicarbonate extractionare separated by chromatography on a silica gel column, eluting withchloroform-methanol (98:2 v/v). Fractions containing the same componentas determined by tlc are combined and further purified by liquidchromatography or preparative tlc followed by recrystallization, to givemore 9α-OH AD plus the compounds of the subject invention.

The mass spectrum of the first eluted compound in its essentially pureform has a molecular ion at 374, and also exhibits intense ions at m/e124, 136 and 137 confirming its close relationship to 9α-OH AD. The irspectrum exhibits bands at 3540 and 3400 cm⁻¹ (hydroxyl) and also at1735 cm⁻¹ and 1655 cm⁻¹ suggesting the presence of two carbonyl groups.Comparison of the ¹ H-nmr spectrum with that of 9α-OH BN acid shows thatthey are virtually identical except for an additional 3 proton peak atδ3.63 where a methyl ester would be expected. This compound is thereforeidentified as the methyl ester of 9α-OH BN acid, and confirmation ofthis is obtained from the ¹³ C-nmr spectrum which shows signals for 23carbon atoms including four methyl groups (δ11.1, 17.0, 19.8 and 5.13),two carbonyls (δ176.9 and 199.0), two olefinic carbons (δ126.7 and168.6) and a quaternary carbon atom bearing oxygen (δ76.2).

The second eluted compound in its essentially pure form is residual9α-OH AD which remains soluble in the acetone-cyclohexane solutiondescribed above.

The third component in its essentially pure form has a molecular weightof 346, the mass spectrum of which again exhibits the characteristicions at m/e 124, 136 and 137. The presence of a hydroxyl group and anunsaturated carbonyl are deduced from infrared peaks at 3400 cm⁻¹ and1650 cm⁻¹ and it is evident from the doublet centered at δ1.05 in the ¹H-nmr spectrum that a side chain similar to that of 9α-OH BN acid and9α-OH BN acid methyl ester is present at C-17. The ¹ H-nmr spectrum indimethyl sulfoxide-d₆ also indicates the presence of both a primary(δ4.18, t, J=5) and a tertiary (δ3.95) alcohol. Signals due to 22 carbonatoms are seen in the ¹³ C-nmr spectrum, including three methyl groups(δ11.1, 16.7 and 19.9), two olefinic carbons (δ126.6 and 169.4), onecarbonyl (δ199.2) and two carbon atoms bearing hydroxyls (δ67.7, tripletand 76.3, singlet). On the basis of the above spectral evidence thestructure 9α-hydroxy-3-oxo-23,24-bisnorchol-4-en-22-ol (9α-OH BNalcohol) is assigned to this compound.

The mass spectrum of the next major eluted compound in its essentiallypure form from this column shows a molecular ion at 304, and the usualintense ions at 124, 136 and 137. Given the evidence close relationshipto 9α-OH AD, and the fact that the ¹³ C-nmr spectrum showed 19 carbonatoms, only one of which was part of a carbonyl group (δ199.3), thiscompound is identified as 9α-OH testosterone, and the structuralassignment is confirmed by comparison with an authentic sample.

EXAMPLE 8

By substituting a microorganism from the genera Arthrobacter, Bacillus,Brevibacterium, Corynebacterium, Microbacterium, Nocardia,Protaminobacter, Serratia, and Streptomyces, in Example 1 forMycobacterium fortuitum ATCC 6842 there are obtained mutantmicroorganisms which are characterized by their ability to selectivelydegrade steroids having 17-alkyl side chains of from 8 to 10 carbonatoms, inclusive, and accumulate the products disclosed herein in thefermentation beer.

EXAMPLE 9

By substituting the mutants obtained in Example 8 for M. fortuitum NRRLB-8119 in Examples 2-7, there are obtained the products as disclosedherein.

EXAMPLE 10

By substituting a microorganism selected from the group consisting ofMycobacterium phlei, M. smegmatis, M. rhodochrous, M. mucosum, and M.butyricum for M. fortuitum ATCC 6842 in Example 1 there are obtainedmutant microorganisms which are characterized by their ability toselectively degrade steroids having 17-alkyl side chains of from 8 to 10carbon atoms, inclusive, and accumulate the products disclosed herein inthe fermentation beer.

EXAMPLE 11

By substituting the mutants obtained in Example 10 for M. fortuitum NRRLB-8119 in Examples 2-7, there are obtained the products as disclosedherein.

The structural formulae for the novel compounds of the invention can beshown as follows:

We claim:
 1. A process for preparing 9α-OH BN acid methyl ester in itsessentially pure form which comprises cultivating Mycobacteriumfortuitum NRRL B-8119 in an aqueous nutrient culture medium underaerobic conditions in the presence of a steroid containing from 8 to 10carbon atoms, inclusive, in the 17-alkyl side chain and isolating saidcompound in its essentially pure form from the culture medium.
 2. Aprocess, according to claim 1, wherein said steroid is sitosterol.
 3. Aprocess, according to claim 1, wherein said steroid is cholesterol.
 4. Aprocess, according to claim 1, wherein said steroid is stigmasterol. 5.A process, according to claim 1, wherein said steroid is campesterol. 6.A process for preparing 9α-OH BN acid methyl ester in its essentiallypure form which comprises cultivating Mycobacterium fortuitum NRRLB-8119 in an aqueous nutrient culture medium under aerobic conditions inthe presence of a mixture of two or more steroids wherein each steroidcontains from 8 to 10 carbon atoms, inclusive, in the 17-alkyl sidechain and isolating said compound in its essentially pure form from theculture medium.
 7. A process, according to claim 6, wherein said steroidmixture is selected from the group consisting of sitosterol,cholesterol, stigmasterol, and campesterol.
 8. A process for preparing9α-OH BN acid methyl ester in its essentially pure form which comprisescultivating a Mycobacterium mutant which is characterized by its abilityto selectively degrade steroids having 17-alkyl side chains of from 8 to10 carbon atoms, inclusive, and accumulate 9α-OH BN acid methyl ester inthe fermentation beer, in an aqueous nutrient culture medium underaerobic conditions in the presence of a steroid containing from 8 to 10carbon atoms, inclusive, in the 17-alkyl side chain and isolating saidcompound in its essentially pure form from the culture medium.
 9. Aprocess, according to claim 8, wherein said mutant microorganism iscultivated in an aqueous nutrient culture medium under aerobicconditions in the presence of a mixture of two or more steroids whereineach steroid contains from 8 to 10 carbon atoms, inclusive, in the17-alkyl side chain and isolating said compound in its essentially pureform from the culture medium.
 10. A process, according to claim 8,wherein said steroid is selected from the group consisting ofsitosterol, cholesterol, stigmasterol, and campesterol.
 11. A process,according to claim 9, wherein said steroid mixture is selected from thegroup consisting of sitosterol, cholesterol, stigmasterol, andcampesterol.